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| Code Block |
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Nextera adaptor style:
5'-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGT-3'
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3'-CAGAGCACCCGAGCCTCTACACATATTCTCTGTC-5'
TruSeq truncated adaptor:
5'-ACACTCTTTCCCTACACGACGCTCTTCCGATCT-3'
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3'-CACTGACCTCAAGTCTGCACACGAGAAGGCTAGA-5'
Nextera adaptor style, with primers overlaid:
First cycle:
3'-GGCTCGGGTGCTCTG CAAAGC TAGAGCATACGGCAGAAGACGAAC-5'
5'-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGT-3' <UNKNOWN> 5'-CTGTCTCTTATACACATCTCCGAGCCCACGAGAC-3'
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3'-CAGAGCACCCGAGCCTCTACACATATTCTCTGTC-5' <UNKNOWN> 3'-TGACAGAGAATATGTGTAGACTGCGACGGCTGCT-5'
Second and subsequent cycles:
AATGATACGGCGACCACCGAGATCTACAC ATCACG TCGTCGGCAGCGTC
3'-AGCAGCCGTCGCAGTCTACACATATTCTCTGTCA <UNKNOWN> 3'-TGACAGAGAATATGTGTAGACTGCGACGGCTGCT-5' |
Cautions, common mistakes, and lessons learned from failure
- Assembling the P7 side adaptor or primer wrong - the key thing to note is that the "cannonical designs" are shown 5' to 3' across the entire finished sequencing construct. So if you're designing a reverse primer for the P7 side you have to use the reverse complement of ALL 3 DESIGN ELEMENTS (flow cell binding site, barcode, and sequencing primer site) and make sure they're in the right order.
- Incorrect P5 dual-index design - the "ACAC" motif in the single index design MUST be repeated on both sides of an index within P5 - see the "dual index" designs specifically.
- Reverse complement barcode sequences in either P5 or P7 side indexes, especially from amplicons - the fact that the Illumina sequencers read i5 differently is a pain - pay attention to that when submitting barcode sequences that will wind up in a sample sheet. And remember that the i7 index is read "forward, top strand" of the canonical design, which is reverse complement of the sequence that appears in a reverse primer used when creating a library.